Our study aims at comparing in C57/Bl male mice, the impact of repeated injections of baclofen (an agonist of GABAB receptor) or diazepam  administered during the alcohol-withdrawal period on hippocampus-dependent memory impairments and brain regional glucocorticoid dysfunction after a short (1-week) or a long (4-week) abstinence. Hence, mice were submitted to a 6-month alcohol consumption (12%v/v) and were progressively withdrawn to water. Then, after a 1- or 4-weeks abstinence, they were submitted to a contextual memory task followed by measurements of corticosterone concentrations in the dorsal hippocampus (dHPC), the ventral hippocampus (vHPC) and the prefrontal cortex (PFC). Results showed that 1- and 4-week withdrawn mice exhibited a severe memory deficit and a significant abnormal rise of the test-induced increase of corticosterone (TICC) in the dHPC, as compared to water-controls or to mice still under alcohol consumption. Repeated daily systemic administrations of decreasing doses of diazepam (ranged from 0.5 to 0.12 mg/kg) or baclofen (ranged from 1.5 to 0.37 mg/kg) during the last 15 days of the withdrawal period, normalized both memory and TICC scores in the dHPC in 1-week withdrawn animals; in contrast, only baclofen-withdrawn mice showed both normal memory performance and TICC scores in the dHPC after a 4-week withdrawal period. In conclusion, the memory improvement observed in 4-week withdrawn mice administered with baclofen stem from the protracted normalization of glucocorticoid activity in the dHPC, a phenomenon encountered only transitorily in diazepam-treated withdrawn mice. Evidence in humans and rodents have shown that alcohol-withdrawal (AW) markedly affects memory linked to hippocampal or prefrontal cortex (PFC) functional disorders and the hypothalamic-pituitary-adrenal axis (HPA) activity –7. Even A way to reduce the HPA axis hyper-activity in abstinent subjects is to act on the GABAergic neurotransmission. Indeed, GABAergic neurons and GCs receptors (GR) have been found to be co-localized in the paraventricular nucleus of the hypothalamus, which demonstrates a critical importance to control the HPA axis activity through the GABAergic mediation –18. BaclAs regards this issue, we implemented a mice model of long-lasting AW-induced GCs and cognitive dysfunction , 14. We The present study is a continuation of our previous work and aims to compare the relative efficacy of diazepam and baclofen in counteracting the persistent hippocampal-dependent memory and brain regional GCs disorders observed after AW in mice. To probe this issue, we first investigated the effects of a 1-week, (1-W) or 4-week (4-W) AW periods on memory in a serial contextual memory task known to involve the dHPC activity , 41. Add2), in a temperature-controlled colony room (22 ± 1°C), under a 12:12 light-dark cycle (lights on at 7:00 a.m.). All test procedures were conducted during the light phase of the cycle between 8.00 and 12.00 a.m. During the food deprivation phase, mice (28 to 32 g) were housed individually and were maintained at 85–90% of their ad libitum body weight throughout the behavioral study. In both experiments 1 and 2, animals were daily handled 6 min/day during the week preceeding the beginning of the behavioral phase, to reduce the emotional reactivity to he experimenter.This study has been conducted on male C57BL/6 mice . Mice were 3 months old upon arrival. They were housed by groups of 10 in collective cages  enclosed with gray Plexiglas walls. On the floor, 4 holes opening on a food cup (3 cm diameter × 2.5 cm in depth) were located 6 cm away from the sidewalls. Photocells located inside each hole quantified automatically the number of head-dips.The contextual and serial memory task (CSD) has been described in full in previous papers , 42, 43 All mice were first food-deprived before being submitted to the behavioral test. The food deprivation procedure is aimed at increasing the motivation for the pellets used as reinforcing agents in the hole-board. To that aim, mice were given a limited quantity of food for 3 consecutive days  before the beginning of the behavioral experiment. This food regimen induced a progressive weight loss comprised between 10 and 12% of the initial weight (before the food deprivation procedure). During this period, some pellets used in the behavioral test were placed in their cage in order to get them used to eating them.During the acquisition phase, food-deprived mice learned two successive discriminations  performed on two different floors differing by the color and texture (white and smooth or black and rough). The floors are alternated from one mouse to another and from one discrimination to another in order to avoid a bias related to their positioning in the series. The two serial discriminations were separated each by a 2-min time interval during which the mouse was placed in its home cage. For D1 and D2, ten 20-mg pellets  were available only in a specific hole out of the 4 holes of the device; the baited holes at D1 and D2 of the acquisition phase were changed from one mouse to another but in all cases, the baited holes at D1 and D2 were systematically opposite and symmetrical.The retention phase occurred 24 h later, mice were replaced for 6 min on the floor used specifically at D1, with no food pellets in the apparatus. We previously showed that the memory of D1 was dependent on the dHPC activity but not on vHPC or PFC ones , 43, 44.2) with continuous access to alcohol. Diazepam  and baclofen  were diluted in a vehicle solution (0.9% NaCl) and injected intraperitoneally . Drug doses were based on pilot experiments and previous studies  during a brief 30-s inhalation exposure followed by a rapid decapitation. The choice of isoflurane relies on the fact that studies have shown that this anesthetic has little or no effect on plasma corticosterone levels in male rats . The homogenized tissues were sonicated by ultra sounds on ice-cold  and centrifuged at 1,300 g for 30 min at 4°C. Supernatants were kept and stored at −80°C until quantification. Samples were prepared  for colorimetric evaluation in a spectrophotometer . Corticosterone was measured using a commercial ELISA assay .Samples (20 ul) of blood were proceeded for blood alcohol concentration using an EnzyChrom™ Ethanol Assay Kit  according to the manufacturer's instructions.hoc tests (Bonferroni/Dunnett). Correlation analyses were performed by the Spearman's correlation coefficient R. For all tests, p < 0.05 was considered statistically significant.Data were expressed as Means ± SEM. Analyses were performed using the Statview 5.0 software . The data were analyzed using one or two-ways ANOVA to determine main factor effects and their interaction, and followed by appropriate post F = 1.02; p = 0.14]. Thus, exposure to alcohol was considered as equivalent among the withdrawn groups. In comparison, the mean daily water consumption was measured in two groups of water controls; the mean daily water consumption was 3.1 ± 0.6 mL which did not significantly differ from alcohol-treated groups (p < 0.09). The blood concentration of ethanol was quantified with a commercial Elisa kit  and was 0.49 ± 0.24 g/L (10.7 ± 5.2 mM or 0.62 ± 0.30%) in mice still under the alcohol regimen in experiment 1 and below the limit of quantification in all withdrawn groups (0 ± 0 g/L) of the study at the time of memory testing.At the end of the feed deprivation period, the weights of the animals ranged from 27.3 ± 0.8 g (minimum) to 31.7 ± 0.6 g (maximum); all mice showed a weight loss of 10% to 12% of their initial weight. Among the withdrawn groups submitted to the CSD tasks, the mean daily alcohol consumption (mL) over the 6-month alcohol exposure was 3.85 ± 0.42 mL/mouse and no significant between-groups difference was observed  and second discriminations . No significant between-group difference was observed on the % number of head-dips in the baited hole of the first and second discrimination .ANOVA's analyses did not evidence a significant between-groups difference on the total amount of head-dips both on the first .No significant between-group difference was observed on the total number of head-dips  whereas 1- and 4-W-withdrawn groups showed a negative one  = 0.019; p = 0.89). Both 1- and 4-W withdrawn mice differed significantly from Alcohol and Water-control groups .The discrimination index was significantly different among the groups , the vHPC  and the PFC .The basal corticosterone concentration was measured in naïve mice from the four cohorts left undisturbed in their home cage during behavioral testing. Corticosterone concentrations are expressed in ng/mL in Since no significant difference was observed in basal corticosterone concentrations in the naïve condition, the test-induced increase of corticosterone concentrations (TICC) was expressed for each group as a percent variation of the naïve condition [100 × (Test - Naive)/Naive].F = 7.72; p = 0.0006] (p = 0.0002). In contrast, TICC scores were significantly increased in 1W-withdrawn (+574.68 ± 103.9 %) and 4W-withdrawn groups (+505.07 ± 90.9%) as compared to Water-controls  and to the Alcohol group .A significant between-group difference was observed [ 0.0006] . The AlcF = 2.31; p = 0.09]. Both the Water-control and Alcohol groups exhibited comparable TICC scores  which were however higher in 1W-withdrawn (+445.5 ± 86.8%) and 4W-withdrawn groups (+396.14 ± 80.5%).ANOVA analyses evidenced no significant between-group difference . Alcohol mice (+295.18 ± 20.91%) exhibited a higher TICC score as compared to Water-controls (+192.5 ± 23.35%) which was increased in 1W- and 4W-withdrawn groups .No significant between-group difference was observed .Data are depicted in F=32.36; p < 0.0001), drugs  = 9.03; p < 0.0006) and the interaction between groups and drugs was also significant . Vehicle-withdrawn mice exhibited a lower discrimination index (-16.20 ± 7.3) as compared to vehicle-Water-controls . Both 1-W-withdrawn diazepam (+29.37 ± 8.86) and 1-W-withdrawn baclofen (+35.04 ± 2.30) groups exhibited higher discrimination indexes as compared to vehicle-withdrawn mice . In contrast, they did not statistically differ from vehicle-Water-controls . In water-controls, diazepam (+46.51 ± 8.93) or baclofen (+46.71 ± 3.38) did not modify the discrimination index as compared to the vehicle-Water-control group .A two-way ANOVA showed significant effects of groups  = 145.5; p < 0.0001], drugs  = 57.19; p < 0.0001] and the interaction between groups and drugs was also significant . Vehicle-withdrawn mice exhibited a lower discrimination index as compared to vehicle-Water-controls . Baclofen-withdrawn mice exhibited a higher discrimination index (+69.08 ± 5.79) as compared to vehicle-withdrawn mice (p < 0.0001) which was not observed in diazepam-withdrawn mice . Baclofen-withdrawn mice did not statistically differ from vehicle-Water-controls (p = 0.052) but significantly differed from diazepam-withdrawn mice (p < 0.0001); in contrast, diazepam-withdrawn mice still exhibited a significant lower discrimination index as compared to vehicle-Water-controls (p < 0.001). Diazepam and baclofen Water-control groups  did not statistically differ from vehicle ones .A two-way ANOVA showed significant effects of groups .Data are expressed in Since no significant difference was observed in basal corticosterone concentration in the naïve condition, the test-induced increase of corticosterone concentrations (TICC) was expressed for each group as a percent variation of the naïve condition [100 × (Test - Naive)/Naive].F = 12.34; p < 0.0001], drugs  and the interaction between groups and drugs was also significant .Data are depicted in F = 0.40; p = 0.52], but a significant effect of drugs  and the interaction between groups and drugs was also significant . More specifically, vehicle 1-W-withdrawn mice (+394.6 ± 46.6%) exhibited a significant increase of TICC scores as compared to respective Water-control mice . Diazepam-1-W-withdrawn mice exhibited a significant reduction of TICC scores as compared to vehicle-withdrawn mice ; a similar reduction of TICC scores was also observed in baclofen-treated withdrawn mice .In the 1-Week groups, a two-way ANOVA showed no significant effects of groups , drugs , and the interaction between groups and drugs was also significant . More specifically, Vehicle 4-W-withdrawn mice exhibited a significant increase of the TICC scores as compared to respective Water-controls  which was significantly lowered in baclofen (+175.7 ± 29.01%) but not in diazepam-treated withdrawn groups . In addition, baclofen-withdrawn mice differed significantly from diazepam-withdrawn ones (p < 0.0001).In the 4-week groups, a two-way ANOVA showed significant effects of groups [withdrawn-4-W and vehicles-4-W; p < 0.0001). More specifically, the higher is the discrimination index, the lower is the TICC score.ANOVA analyses performed in all groups within Experiment 2 showed a significant negative correlation between the discrimination index and the TICC score . Interestingly, as shown in The discrimination index correlated negatively with TICC scores . Interestingly, as shown in The discrimination index correlated negatively with TICC scores  or a long (4-W) abstinence from alcohol. Results showed that both withdrawn groups exhibited contextual memory deficits and significant abnormal rise of the test-induced increase of corticosterone specifically in the dHPC compared to water controls. Interestingly, while both chronic diazepam or baclofen treatment during the withdrawal period rescued contextual memory deficits and prevented the test-induced GCs alterations in 1-W withdrawn animals, only baclofen-treated animals showed memory performance and test-induced rise of corticosterone similar to those of water-controls after a 4-Week withdrawal period.A prime issue to be addressed rested with assessing whether the memory and endocrine alterations observed in alcohol-withdrawn mice may be caused by differences in diets. Findings evidenced that differences in the daily amount of food consumption may not be held accountable for the deficits since we have already demonstrated that pair-fed animals receiving an isocaloric solution of dextri-maltose during the same duration (6 months period) of alcohol exposure exhibited no memory deficits . MoreoveMoreover, it has been already reported that GABAB and GABAA receptor agonists modulate alcohol drinking , 59 and In the present study, we did not directly measure locomotor activity in both the acquisition and test phases of the task. It is known that alcohol consumption and withdrawal may induce side effects such as an alteration of locomotor activity, even though contrasting data are reported according to the duration of the treatments and withdrawal period (see in 60). However, since alterations in locomotor activity can potentially alter the performance in the memory task, one cannot exclude that the lack of measurement of this parameter may be a limitation to the study. Nevertheless, substantial data allow us to rule out this hypothesis. First, we did not observe in the present study a significant difference in the exploration of the total number of head-dips in the baited and unbaited holes among the groups during the acquisition phase: such a finding infers that locomotor activity is similar in all groups. This observation confirmed previous experimental data showing transitory impairments in exploring a 9 hole-board only in 1-week but not in 6-weeks withdrawn mice, as compared to controls . In addiOur present findings agree with other studies showing that AW produced sustained alterations of memory in alcohol-withdrawn rodents as compared to water-controls or to rodents still under alcohol regimen , 13, 43.From a psychological point of view, exaggerated anxiety levels resulting from alcohol-withdrawal have been considered as a potential key factor of the long-lasting maintenance of cognitive deficits over time. Indeed, human and rodent studies have reported enhanced anxiety-like behaviors in various tests during ethanol withdrawal . In our Endocrine studies in humans , 2, 4, 6Interestingly, the vHPC and the PFC exhibited non-significant enhancements of the TICC scores as compared to water-controls. We previously showed that these brain areas are not recruited in the processes sustaining memory of the first discrimination in the CSD task , 44, 67.In the present study, diazepam and baclofen were undetectable in the blood of animals at the time of behavioral testing  and therWe reported here-above that repeated diazepam administrations counteract only transitorily the memory impairment and neuroendocrine disorders in 1-W-withdrawn but not in 4-W-withdrawn mice. We previously evidenced corrective effects of a similar repeated diazepam administration on working memory alterations after a short (1-week) but not a long (6-weeks) withdrawal period . The faiIn sharp contrast, baclofen suppressed the protracted memory impairments and normalized the test-induced increase of corticosterone in the dHPC regardless the withdrawal periods, showing thereby prolonged counteracting effects of baclofen on these impairments. Interestingly, the beneficial effects of baclofen on alcohol addiction and relapse in abstinent subjects as well on HPA axis disorders and protracted cognitive impairments have been well substantiated , 39, 82.Since several studies evidenced different HPA axis responses to alcohol consumption and withdrawal in female as compared to male rodents , 85, ourOverall, the present study provides evidence that acting on the GABAB receptor through repeated baclofen administration during the alcohol-withdrawal phase counteracted the persistent hyper-reactivity of HPA axis to behavioral testing in the dHPC and rescued memory found to be altered up to 4-weeks after the cessation of alcohol intake; in contrast, diazepam, a compound having an agonist action on the GABAA receptor, induced only a transitory beneficial effect on the memory and endocrinal alterations.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by EU Directive 2010/63/EU and by the local Ethical Committee of Bordeaux (#5012089).HN and MF were equally involved in behavioral and neuroendocrine studies. MN, CP, and BD were involved in data and statistical analyses, the experimental design, and writing of the article. All authors have approved the final article.frances.ash@orange.fr).This research was supported by a grant from FRA  and the CNRS. We thank Dr. Frances Ash for language proofreading (The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lactobacillus plantarum (L. plantarum), litchi pericarp oligomeric procyanidins (LPOPCs) were bioconverted to several phenolic acids, which increased the antioxidant activity as expected. However, antiglycation ability and trapping carbonyl compounds capacity both weakened and it might be the primary reason for decreasing the inhibitory effect on AGE formation. Furthermore, it was found that LPOPCs incubated with L. plantarum inhibited the activity of digestive enzymes and thus decreased the digestibility of glycated protein. Our study systematically proposed for the first time that procyanidins bioconversion is an effective means to improve the antioxidant activity but has no remarkable promoting effect on AGEs inhibition.Lactic acid bacteria (LAB) have already been used as fermentation strains to enhance the antioxidant capacity of polyphenols. Antioxidant capacity is one of the most important factors to inhibit advanced glycation end product (AGE) formation and could LAB increase the inhibitory capacity of procyanidins on AGEs formation? It was surprising that opposite results were obtained both in simulated food processing and gastrointestinal digestion systems. After incubation with  Advanced glycation end products (AGEs), such as carboxymethyl lysine (CML) and carboxyethyl lysine (CEL), are generated by the reaction between reducing sugars and the free amino residues of proteins, lipids, or nucleic acids . Dietaryin vitro , such as  of food –10. In a of food .L. plantarum through galloyl-esterase, decarboxylase, benzyl alcohol dehydrogenase, and tannase  were obtained from Guangzhou, China and arrived in the laboratory within 24 h postharvest. The fruits were peeled immediately, and the pericarp was kept in sealed polyethylene bags at −18°C prior to extraction for 3 days. L. plantarum used in this study was purchased from the China Center of Industrial Culture Collection. (–)-Epicatechin (EC), (+)-catechin (CC), 3,4-Dihydroxyphenylacetic acid , 4-hydroxyphenylpropionic acid (4-HPA), m-coumaric acid (m-CA), p-coumaric acid (p-CA), acetonitrile, methanol, and acetic acid were from Sigma Chemical Co. . Formic acid and 2,4,6-tris(2-pyridyl)-s-triazine were obtained from Aladdin . CML was purchased from Toronto Research Chemicals Inc. . All other chemicals were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. of China .Fruits of litchi (The oligomeric procyanidins of litchi pericarp (LPOPCs) were obtained according to the method by Li et al. . BrieflyLactobacillus plantarum was inoculated in de Man, Rogosa and Sharpe (MRS) medium at 37°C for 18 h, then diluted to obtain the concentration of approximately 107 cfu/ml. The bacterial suspension was mixed with LPOPCs at different concentrations and maintained at 37°C for 48 h under aerobic conditions. The growth was measured at 2 h intervals by assessing optical density at 600 nm (OD600) using a UV-1601 spectrophotometer .Glucose (42 mM), lysine (42 mM), and sodium azide (3 mM) were used as a glucose-lysine model heating at 50°C for 5 days.3 and thereupon mixed with 2 ml of simulated intestinal fluid . The intestinal digestion continued in vitro for 2 h at 37°C and was quenched in boiling water bath for 3 min.Glycated bovine serum albumin (BSA) was prepared using the method described by Zhao et al. . GlycateL. plantarum and non-inoculated LPOPCs were used as control.Fluorescent AGEs and CML were determined according to methods by Wu et al. . L. planA liquid chromatography/electrospray ionization with multi-stage mass spectrometry  was used to detect the composition and structure of phenolic acids after fermentation . A ZORBA3+-2,4,6-tripyridyl-s-triazine [TPTZ]) to the ferrous form (Fe2+-TPTZ), followed by spectrophotometric analysis  were used for calibration.The assay was based on the reduction of a ferric 2,4,6-tris(2-pyridyl)-1,3,5-triazine  and measured at a fixed 173° scattering angle in 3.5 ml of plastic cuvettes.The enzyme activity was detected by the method of Zhu et al. . The incTrypsin activity was assayed by utilizing N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPNA) as substrate . In the p < 0.05). The graph was drawn by OriginPro 8.0.All experiments were performed in triplicate and reported as mean ± standard deviation (SD). Significant differences between mean values were determined by one-way ANOVA followed by Duncan's multiple range tests and confirmed the results at 95% confidence interval  are listed in m-CA, and p-CA. LPOPCs could be bioconverted by L. plantarum to the small molecules by some inducible enzymes, such as tannase, which possess the ability to hydrolyze the ester and depside linkages in wide-ranging substrates, such as gallotannins, epigallocatechin-3-gallate, gallic acid esters, and EC gallate, to release gallic acid and glucose  all had lower antiglycation, antioxidant, and α-dicarbonyl compound scavenging capacities , the Collaborative Grant-in-Aid of the HBUT National 111 Center for Cellular Regulation and Molecular Pharmaceutics (No. XBTK-2021003), Key Laboratory of Green Processing and Intelligent Manufacturing of Lingnan Specialty Food, Ministry of Agriculture, and the Open Research Fund Program of Key Laboratory of Cleaner Production and Integrated Resource Utilization of China National Light Industry (CP-2020-YB6).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Lactobacillus plantarum (LP) KFY02 on low-fiber diet-induced constipation in mice. LP-KFY02 was isolated from the natural fermented yogurt in Korla of Xinjiang. The mice with low-fiber diet-induced constipation in experimental groups were administered 1 × 109 CFU/kg LP-KFY02 (KFY02H) and 1 × 108 CFU/kg LP-KFY02 (KFY02L). After LP-KFY02 treatment with constipation mice, the mice fecal water content, intestinal transit ability and defecation time of constipated mice were improved. The mice fecal flora diversity, abundance and structure of the intestinal flora were regulated to the balanced state. The mice serum levels of gut motility related neuroendocrine factors have been increased, the intestinal mucosal barrier function and gut motility related gene expression were regulated in mice colon tissues. At the same time, the mice colon tissue damage were improved. These parameters in the KFY02H group were close to the normal group. These results suggested that LP-KFY02 could be considered as a potential probiotic to help alleviate low-fiber diet-induced constipation. They also provided a theoretical basis for the study of probiotics to relieve constipation by regulating intestinal flora.This study aimed to examine the ameliorating effect of  Constipation is a high-incidence disease worldwide, the increasing global prevalence of the symptoms of constipation adversely affects the quality of life of symptomatic patients . With thFC is intractable constipation characterized by colonic dysfunction and conduction abnormalities resulting in weakened intestinal transit capacity. It can even cause a series of serious complications . The resBifidobacterium, Lactobacillus, and so on) and the increase in the abundance of potential pathogenic bacteria   . It is cd so on) . The maid so on) . (2) Thed so on) . At the d so on) . Transied so on) . (3) Thed so on) . C-kit id so on) . At the d so on) . TherefoThe Xinjiang Uygur Autonomous Region is located in the frontier area of southwestern China, and the livestock industry is the local pillar industry. Many ethnic minorities reside in Xinjiang, and most of them have the habit of drinking dairy products. Their homemade natural fermented yogurt is one of the most common dairy products . NaturalTherefore, this study explored the improvement effect of lactic acid bacteria isolated from Xinjiang natural fermented yogurt on a low-fiber diet-induced constipation model. Based on the findings of this study, we will explore safe and effective microbial resources that can improve constipation and provide research data support for methods to improve FC by regulating intestinal flora disturbance.Lactobacillus plantarum (LP) KFY02 was isolated from the natural fermented yogurt in Korla of Xinjiang Uygur Autonomous Region. The strain was preserved at the China General Microbiological Culture Collection Center  with a preservation number CGMCC no. 15638. Lactobacillus delbrueckii subsp. Bulgaricus NQ2508 (LB) was obtained from the China Center of Industrial Culture Collection (preservation number: CICC 6047).The experimental strain ad libitum access to standard chow and water. After 7 days of adaptive feeding, the mice were randomly and equally divided into five groups: normal, model, positive control, KFY02H group: high-dose LP-KFY02, KFY02L group: low-dose LP-KFY02, with 10 mice per group (five mice per cage). The experimental procedure is shown in 9 CFU/kg LP-KFY02, the mice in the KFY02L group were administered 1 × 108 CFU/kg LP-KFY02, and the mice in the positive control group were administered 1 × 109 CFU/kg LB per day for 14 days. During the experiment, the mice body weight were recorded per week.Fifty 6-week-old male Institute of Cancer Research (ICR) mice were obtained from the Chongqing Medical University, Chongqing, China [SCXK (YU) 2017-0001]. The mice were raised in a climate-controlled room  on a 12 h light/dark cycle (lights on at 7 a.m.) with On the 35th day, all mice were fasted for 16 h and then given activated carbon solution (10 mL/kg BW). In each group, five mice were observed to record the time of the first black stool, and the remaining five mice were used to measure the gastrointestinal transit rate. The gastrointestinal transit rate of activated charcoal was calculated according to the following equation:Gastrointestinal transit rate (%) =  × 100%For measuring mice fecal water content, the fresh mice stools were collected and weighed on 22nd and 35th day, and the dry weight was obtained by drying fresh stools in an oven at 70°C for 48 h. The mice fecal water content was calculated according to the following equation:Fecal water content (%) = [(fresh feces weight - dry feces weight)/fresh feces weight] × 100%For microbial analysis, the fresh mice feces were collected in individual sterile eppendorf tubes on 35th day, then stored at –80°C for further deoxyribonucleic acid (DNA) extraction.At the end of the experiment, the mice were sacrificed by cervical dislocation. The blood from the mice were centrifuged at 4°C and 4,000 rpm for 10 min to collect the serum. The colon tissues were collected and stored at –80°C for further experiments.1Genomic DNA of mice feces were extracted using a FastPrep DNA extraction kit , and the extracted genomic DNA was detected by 1% agarose gel electrophoresis. The polymerase chain reaction (PCR) amplification and sequencing region was 338F_806R, and the amplification primers used universal primers (338F/806R) of the 16S rRNA gene V3–V4 region. The primer sequences were 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). PCR formal experiments used a TransGen AP221-02: TransStart Fastpfu DNA Polymerase, 20-μL reaction system. Reaction conditions: 95°C, 10 min, 95°C, 15 s, 58°C, 30 s, 72°C, 30 s, 40 cycles. DNA extraction, PCR amplification, and sequencing were done in collaboration with Shanghai Majorbio Bio-pharm Technology Co., Ltd. The 16S rRNA sequence data were analyzed using the QIIME2-pipeline on the Majorbio Cloud Platform.The serum levels of MTL, SP, gastrin, VIP, and 5-HT were quantified using ELISA kits following the manufacturer’s protocols .A portion of mice colon tissues were cut and immersed in 10% formalin solution. Paraffin-embedded sections were made and stained with hematoxylin and eosin in collaboration with Wuhan Servicebio Technology Co., Ltd. The histological sections were observed under a biological microscope .CT–ΔΔ method with GAPDH as the internal reference gene , the RNA was reverse transcribed into cDNA and then amplified using a Real-Time PCR System . The relative mRNA expression of each target gene SCF, c-kit, transient receptor potential vanilloid 1 (TRPV1), glial cell line–derived neurotrophic factor (GDNF), claudin-1, and occludin were calculated using the formula 2p < 0.05 indicated a statistically significant difference.Data were presented as the mean ± standard deviation. The SPSS 19.0 software  was used to conduct the analysis. Differences between the mean values for individual groups were assessed with a one-way analysis of variance and Duncan’s multiple range tests. A p < 0.05). Among them, the body weight of mice in the model group was significantly lower than the KFY02H group, KFY02L group, and the LB group (p < 0.05).In order to evaluate the effect of LP-KFY02 on the body weight change of constipated mice, the mice body weight were measured per week . The bodp < 0.05), indicating that the constipation model was successfully established. On 35th day of constipation induction, after gavage with LP-KFY02, the fecal water content of mice increased significantly compared with the model group (p < 0.05).The fecal water content can evaluate whether the mice constipation model is successfully induced. Dry stools are common in constipated patients. An increase in fecal water content indicates an increase in intestinal peristalsis, thereby relieving the symptoms of constipation. As shown in p < 0.05).The excretion time of the first black stool in mice is also one of the important indicators for judging the intestinal peristalsis ability of constipated mice. Excessive time to first black stool indicates severe constipation symptoms. As shown in p < 0.05), KFY02H group was close to the LB group, which indicating that LP-KFY02 can promote intestinal motility.The gastrointestinal transit rate can directly reflect the intestinal motility. In p < 0.05). At the same time, the sobs index, chao index, and shannon index were significantly higher in the KFY02H group compared with the other groups (p < 0.05). The simpson index of KFY02H group was 0.04 ± 0.01, which was significantly decreased compared with the other groups (p < 0.05). The alpha diversity results showed the higher community richness and bacterial diversity of constipated mice feces in the KFY02H group.The alpha diversity in mice feces was shown in via principal component analysis (PCA). In The beta diversity was determined Firmicutes and Bacteroidetes, followed by Actinobacteria, Proteobacteria, Desulfobacterota, Spirochaetota, Deferribacteres, Patescibacteria, and Campilobacterota. Following the statistical results, in the model group, the Firmicutes abundance increased significantly, the Bacteroidetes abundance decreased significantly compared with the other groups (p < 0.05). However, in KFY02H group, the Firmicutes abundance decreased significantly, the Bacteroidetes abundance increased significantly compared with the model group, the LB group and the KFY02L group (p < 0.05), which is close to the normal group.The composition of the gut microbiota flora at the phyla level and genus level were explored. As shown in Lactobacillus, norank_f_Muribaculaceae, and Bacteroidetes abundance significantly increased in the KFY02H group (p < 0.05), which is close to the normal group. These results indicated that composition of the gut microbiota flora in the KFY02H group was similar to the normal group. Therefore, LP-KFY02 maintained the gut microbiota of mice to a balanced state.The composition of the gut microbiota flora at the genus level in the five groups is shown in p < 0.05). After treatment with LP-KFY02 and LB, the serum levels of MTL, SP, GAS, VIP, and 5-HT increased significantly, which were close to the level in the normal group (p < 0.05). The levels in KFY02H group were the closest to those in the normal group.As shown in As shown in p < 0.05). In contrast, the expression levels of these markers were downregulated in the model group. Also, the expression of TRPV-1 was downregulated in the KFY02H group, KFY02L group compared with the model group (p < 0.05). The mRNA expression levels in the KFY02H group was similar to that in the normal group.We also evaluated the mRNA expression levels of SCF, c-kit, TRPV1, GDNF, claudin-1, and occludin. Bifidobacterium bifidum G9-1  are specialized epithelial cells distributed in the gastrointestinal mucosal cells. Although their total number is less than 1% of the total intestinal epithelial cells, it is an important carrier to maintain the normal function of the brain–gut axis, and the polypeptide secreted by EEC is a key intermediate transmitter . These pIn the present study, our data showed that LP-KFY02 helped mice with constipation in restoring these neurotransmitter levels to normal that helped relieve constipation. The KFY02H group showed a more pronounced effect. We considered that favorable intestinal flora state mediated by LP-KFY02 influenced the production of neuroendocrine factors and gastrointestinal hormones through the brain-gut axis, thereby regulating intestinal motility.3.Probiotics affect the intestinal mucosal immune system.The intestinal mucosal barrier mainly refers to the digestive tract barrier composed of the constantly renewed intestinal epithelial cells as the structural basis . The intp < 0.05), especially in the KFY02H group, which was similar to that in the normal groups. The expression of these genes is associated with the maintenance of intestinal mucosal barrier function and the regulation of intestinal motility. So we considered that favorable intestinal flora environment mediated by LP-KFY02 avoided the activation of mucosal immune responses and the release of inflammatory cytokines, thereby avoiding intestinal sensory and motor abnormalities caused by impaired intestinal mucosal barrier.Our results showed that LP-KFY02 enhanced the intestinal mucosal barrier function in various ways: (1) After LP-KFY0 treatment the mRNA expression of claudin-1 and occludin were significantly upregulated, thereby regulating tight junctions between intestinal epithelial cells, (2) Through histopathological sections we observed that LP-KFY02 maintained the integrity of gut structure, which avoided constipation induced intestinal barrier damage and intestinal inflammation. (3) Our results also showed that after LP-KFY02 treatment, mRNA expression of SCF, c-Kit, GDNF were significantly upregulated and TRPV1 expression was downregulated significantly (In summary, we studied amelioration effect of LP-KFY02 on low-fiber diet-induced constipation in mice. After LP-KFY02 treatment with constipation mice, the mice fecal water content, intestinal transit ability and defecation time of constipated mice were improved. The mice fecal flora diversity, abundance and structure of the intestinal flora were regulated to the balanced state. The mice serum levels of gut motility related neuroendocrine factors have been increased, the intestinal mucosal barrier function and gut motility related gene expression were regulated in mice colon tissues. At the same time, the mice colon tissue damage were improved. These parameters in the KFY02H group were close to the normal group. These results suggested that LP-KFY02 could be considered as a potential probiotic to help alleviate low-fiber diet-induced constipation. They also provided a theoretical basis for the study of probiotics to relieve constipation by regulating intestinal flora.The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.This animal study was reviewed and approved by the Ethics Committee of Chongqing Collaborative Innovation Center for Functional Food.RY and XZ performed the majority of the experiments and wrote the manuscript. TL and RX contributed to the data analysis. ZY designed and supervised the study and checked the final manuscript. All authors read and approved the final manuscript.


The development of wearable electronic devices has greatly stimulated the research interest of textile-based strain sensors, which can effectively combine functionality with wearability. In this work, the fabrication of highly stretchable and compressible strain sensors from weft-knitted spacer fabric was reported. Carbon nanotubes and polypyrrole were deposited on the surface of fabric via an in situ polymerization approach to reduce the electrical resistance. The as-fabricated WSP-CNT-PPy strain sensor exhibits high electrical conductivity and stable strain-sensing performance under different stretching deformations. The WSP-CNT-PPy strain sensor can be stretched up to 450% and compressed to 60% with a pressure of less than 50 KPa, which can be attributed to the unique loop and interval filament structures. The distinguishing response efficiency of WSP-CNT-PPy can effectively detect faint and strenuous body movements. In addition, the electrochemical behavior of WSP-CNT-PPy was also characterized to study the comprehensive properties. The electro-heating performance was also evaluated for feasible Joule heater applications. This work demonstrates the practicability of WSP-CNT-PPy strain sensor fabric for real-time monitoring in promising wearable garments. In the past few years, wearable electronic devices have gradually emerged as a field of interest, which has received tremendous attention with respectable progress ,2,3,4. AGenerally, knitted textiles can be intrinsically extended in both weft and warp directions due to the loop constructions. In the past decades, fabrication of knitted fabric strain sensors for human body movement detection has been a popular research topic ,27,28,29According to the reported literature, compressible strain sensors were conventionally made of elastic polymer materials. An aerogel sensor consisting of aramid nanofibers was successfully fabricated, which can be used for wearable electronics in detecting pressure with smart shoes and insoles . Pan et 2. Single-walled carbon nanotubes  obtained from Nanjing XFNANO Materials Tech Co., Ltd., China. Pyrrole and Cetyltrimethylammonium bromide (CTAB) exhibited the purity of chemical grade. Sodium dodecylbenzene sulfonate (SDBS), FeCl3·6H2O, phosphoric acid, and anhydrous ethanol were obtained from Pharmaceutical Group Chemical Reagents Co., Ltd., Shanghai, China.Weft-knitted spacer fabric in this study was purchased from Alibaba, Co., Ltd., China. The superficial layer of weft-knitted spacer fabric is polyamide filament with the specifications of 240D/36F, and the interval yarn is polyester filament with a diameter of 60 μm. The thickness of the weft-knitted spacer fabric is 4.0 mm, and the surface area weight is 320 g/mThe WSP was firstly pretreated with an acetone solution for 5 h, dried in natural conditions, then repeatedly soaked in 0.15 wt % single-wall carbon nanotube solutions to obtain stable electrical resistance. After a 12 h period of drying at 70 ℃, the treated sample was denoted as the carbon nanotube weft spacer fabric (WSP-CNT). An amount of 6 mL of pyrrole was poured into 60 mL of deionized water with 0.208 g CTAB and 0.216 g SDBS dispersant under magnetic stirring conditions. An amount of 1 cm × 2 cm WSP-CNT was placed into pyrrole solution for a 2 h ice bath shock. An amount of 0.5 mol/L ferric chloride solution of 60 mL was slowly dropped into the pyrrole solution for an in situ polymerization reaction. The sample was washed with anhydrous ethanol and deionized water three times, and then dried at 70 °C for 12 h to prepare the polypyrrole-deposited WSP-CNT (WSP-CNT-PPy), as shown in The microstructures and surface morphologies of the prepared samples were characterized by scanning electron microscopy . The tensile and compression properties of the samples were measured by Instron equipment. The electrical resistance was measured by an electrochemical workstation of CHI604E, and the corresponding resistance variation by digital multimeter. Ω/cm is the most widely used unit to characterize the surface electrical resistance, therefore, the unit of Ω/cm was used to measure the resistance of the strain sensors. The cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and galvanostatic charge-discharge (GCD) were carried out. The thermal imaging and photo-induced heating performance of the WSP-CNT-PPy strain sensor was characterized by an infrared thermal camera (FLIR ONE Pro), and both the surface temperature and thermal images were recorded. All experiments were performed under the standard temperature of 25 °C and humidity of 65%.The optical images of the WSP strain sensor under both unstretched and stretched in weft direction conditions are shown in The microscopic morphologies of the WSP-CNT and WSP-CNT-PPy strain sensors were characterized by SEM, as shown in Excellent mechanical performance has been considered an important feature in the engineering of wearable applications. The mechanical properties of WSP, WSP-CNT, and WSP-CNT-PPy were studied. In 0 gradually increases with the increase in strain elongation. However, the culmination of the ΔR/R0 curve sags in the middle and it becomes increasingly obvious with higher tensile strain. This phenomenon can be attributed to the inhomogeneity of the knitting loops of weft-knitted spacer fabric under strain conditions. These measured results are identical with the measured values in 0) of the WSP-CNT-PPy is shown in 0 ranging from 0 to 450%. The stages of 0–200% and 200–450% are related to a gauge factor (GF) of 2.226 and 1.607, respectively. The variation in the ΔR/R0 can be attributed to the distortion of the knitted structure due to loading force. Compared with reported studies, this indicates that the WSP-CNT-PPy strain sensor exhibits a large sensing deformation. For instance, Souri et al. [0 also gradually increases.The I–V curves are shown in i et al.  reportedi et al. . The slo0 data with periodic traction and relaxation of different loaded conditions. The result indicates that the WSP-CNT-PPy exhibits a reliable response under various external stimuli, thus ensuring reliability in practical applications.In detail, the strain sensor fabric was initially elongated, and the knitted loops were divided step-by-step. The forced stretching led to the crack of conductive materials and abruption of the deposited nanoparticles. Thus, the sample performance mainly included two steps of 0–200% and 200–450%. Both high sensitivity and wide strain sensing range are important parameters in detecting human motions. The sensitivity of the WSP-CNT-PPy strain sensor to various stimuli conditions was evaluated with the periodic pressure of 10%, and the resulting loading speed was 10 mm/min, 50 mm/min, 75 mm/min, and 100 mm/min, respectively, as shown in 0 value exhibits a constant increase and decrease in every experiment, which demonstrates the robust reproducibility of the strain sensor.In addition, the cyclic stability of the WSP-CNT-PPy was further measured in 0 within 0–0.04 corresponds to shallow breath, while the ΔR/R0 within 0–0.16 corresponds to deep breath. The actual time variation of the ΔR/R0 data with human breath indicates the quick induction and good sensitivity of the WSP-CNT-PPy. 0 responses for finger down and wrist up–down motion, respectively. The results indicate that the spectra are distinctive for different movements. Moreover, the spectra have also exhibited robust repeatability in the continuous up–down process. The ΔR/R0 data cyclically varied corresponding to the movement and resulted in a highly periodical spectra. The tiny movements of the breath, finger, and wrist were reflected by the observed curve of ΔR/R0, which indicates the shrinkage of muscles or skeleton. These measured results indicate that the strain-sensing system is effective to detect various human motions.To further investigate the promising wearable applications of the WSP-CNT-PPy strain sensor, a strain-sensing method to monitor various human movements was established. The WSP-CNT-PPy-based strain-sensing device exhibited high sensitivity in recognition of tiny motions. In 0) of the WSP-CNT-PPy strain sensor when subjected to forced pressure. It can be seen that the curve was divided into four stages: S1 = −0.082, S2 = −0.107, S3 = −0.002, and S4 = −0.00004, respectively. The ΔR/R0 shows a linear decrease against the increasing compressive pressure in different ranges. To investigate the properties in practical applications, the ΔR/R0 responses to repeated compressing and releasing cycles are illustrated in The robust compression capability of fabric has been considered as the foundation to fabricate a compressive strain sensor. Both high compression sensitivity and wide strain range are important parameters for enhancing the sensing efficiency. With the increase in forced pressure, the change of strain is shown in 0 spectrum of WSP-CNT-PPy is recorded, where the loading speed is 1 mm/min, 5 mm/min, 20 mm/min, and 60 mm/min, respectively.The compress–release curves with pressures of 36 Pa, 72 Pa, 144 Pa, and 180 Pa are shown in 0 signal exhibits good reproducibility for at least 10 min. Furthermore, the current–voltage (I–V) curve of WSP-CNT-PPy under different compressive pressure has been studied, as shown in In addition, the cyclic compression stability of the WSP-CNT-PPy was recorded in 0 corresponds to 0 to negative 0.4 with the behavior of compressing and releasing. The ΔR/R0 value with the pressing process indicates good sensitivity properties of the WSP-CNT-PPy compression sensor. 0 related to foot down and up, where the cyclic variation of ΔR/R0 corresponds to zero to negative 0.6. It can be stated that the compressive stress is higher than the finger. 0 curve was cyclically changed due to the compression behavior, thus to obtain a highly periodical spectrum. This indicates that the WSP-CNT-PPy sensor is effective in detecting compression behaviors in different compressive strain-sensing applications.The integrated strain-sensing equipment was taken to evaluate the compression sensing efficiency of WSP-CNT-PPy. The result indicates that the WSP-CNT-PPy exhibited good sensitivity to recognize different compression behavior. In 2), and ΔU is the potential working window (V). The areal capacitance of WSP-CNT-PPy at 1 mV/s is approximately 1765.6 m F/cm2. The GCD data of various areal currents ranging from 0.8 to 2.4 mA/cm2 are presented in gcd = It/(AΔU), the calculated areal special capacitance Cgcd is 740 mF/cm2 at 0.8 mA/cm2, with a capacitance retention of 87.4% when the current density increases seven times. The ED is 65.8 μW h/cm2 and the PD is 320.1 μW/cm2 according to the equation of ED = Cgcd ΔU2/7200 and PD = 3600 ED/t, at a current of 0.8 mA/cm2.Electrochemical sensors have been widely used in various areas due to the advantages of their simplicity and convenience. The preparation of flexible electrochemical sensors is based on fibrous materials and deformability of the material is important to install the devices for consecutive charging–discharging. The electrochemical performance of the WSP-CNT-PPy fabric was evaluated by CV, GCD, and EIS methods. In detail, WSP-CNT-PPy was measured and the I–V curves at different scanning rates are shown in EIS was recorded under the frequency between 0.01 and 100 kHz of 50 mV current, as shown in the Nyquist plot of 2t/R, where E is heat produced by Joule effect, V is the applied voltage, t is time, and R is electrical resistance. Electric heating is the process concerning the energy of electric current converted into heat, which is also referred to as Ohmic heating or Joule heating. Considering the simplicity of electrical power and relative high energy conversion efficiency, electrical heating is the most widely used method for various heating applications. The electro-heating performance of WSP-CNT-PPy was evaluated as shown in In this work, a stretchable and compressive strain sensor with good extensibility and sensitivity was prepared by employing weft-knitted spacer fabric as a matrix. Carbon nanotubes and polypyrrole were successfully deposited on the surface of the fabric through an in situ polymerization process to obtain high conductivity. The unique knitted loop structures together with interval filaments are beneficial to improve strain sensing efficiency under both tiny and high deformations. The WSP-CNT-PPy strain sensor also exhibited excellent electrochemical performance at various strain and pressure conditions. It is suggested that highly stretchable and compressive WSP-CNT-PPy can be used as a potential candidate for future wearable electronic devices. In addition, the as-fabricated WSP-CNT-PPy strain fabric also exhibited robust electrochemical and electroheating properties, and the heating/cooling process exhibits good repeatability. The present work would enable the development of next-generation carbon-nanotube-based wearable textiles for multifunctional applications.


Objective: FCN-159 is a highly active mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor in patients with advanced melanoma and neurofibromatosis type 1 (NF1). We report a population pharmacokinetic (PopPK) model-based analysis of FCN-159 and its application to inform dose selection for NF1 pediatric trials.Methods: PK data collected from patients with advanced melanoma and NF1 in two clinical studies (NCT03932253 and NCT04954001) were analyzed using a non-linear mixed effects model. The adult model was adapted by incorporating allometric scaling for PK projection in 2–17 years old children. Pediatric exposure in different body surface area (BSA) bins was simulated to identify nominal doses  and BSA bin cutoffs to achieve exposure comparable to adults’ optimal exposure across the entire pediatric BSA range.Results: The final dataset consisted of 45 subjects with a total of 1030 PK samples. The PK of FCN-159 was well-described by a 2-compartment model with first-order linear elimination and delayed first-order absorption. Covariates, including BSA, age, sex, albumin, total protein, and cancer type, were identified as statistically significant predictors of FCN-159 disposition. Simulations based on the final model projected daily doses of 4 mg/m2 QD with optimized BSA bin cutoffs would allow fixed nominal doses within each bin and result in steady state exposure approximating the adult exposure observed at the recommended phase 2 dose (RP2D) in NF1, which is 8 mg QD.Conclusion: The developed population PK model adequately described the PK profile of FCN-159, which was adapted using allometric scaling to inform dose selection for NF1 pediatric trials. Rat sarcoma virus (RAS) oncogene mutations and abnormal activation play an essential role in the development of melanoma and neurofibromatosis type 1 (NF1) . ActivatMEK inhibitors selectively inhibit mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) activity in individuals with v-raf murine sarcoma viral oncogene homolog B1 (BRAF) or RAS-positive malignancies or NF1, and impair signal transduction pathways that regulate proliferation and survival in cancer cells . SeveralFCN-159 is a highly active MEK1/2 inhibitor that inhibits MEK phosphorylation with low inhibitory potential for other kinases . The molvia multiple pathways, primary by cytochrome P450 (CYP) 3A4 and to a lesser extent by CYP2C19 and CYP2C8  and the guiding principles of the Declaration of Helsinki. All study participants provided signed informed consent before enrollment.FCN-159 plasma concentrations from 2 phase I clinical trials, including FCN-159-001 (NCT03932253) and FCN-159-002 (NCT04954001), were collected and pooled in the PopPK analysis . FCN-159In these studies, FCN-159 plasma concentrations were analyzed by a validated liquid chromatography-tandem mass spectrometry (LC-MS-MS) method with an assay range of 0.2–200 ng/ml.http://www.r-project.org/). Population PK analyses and all related simulations were performed with NONMEM version 7.4  and Perl-speaks-NONMEM version 5.0.0 . NONMEM was compiled based on GNU Fortran (GCC) 4.6.3 and was called by Strawberry Perl (64-bit) 5.28.1.1 and Perl-speaks-NONMEM 4.8.1.Analysis data assembly, descriptive statistics, and plots were programmed using R version 4.0.3  and volume(s) of distribution. The first-order conditional estimation method with interaction (FOCE-I) was used for parameter estimations. The base PopPK model was selected among candidate models by comparing the Akaike information criterion (AIC), goodness-of-fit, plausibility of parameter estimates, precision of parameter estimates, minimum objective function value (OFV), and eta shrinkage values. The two-compartment model with first-order elimination and first-order absorption with lag time was selected as the structural model. The residual variability was modeled as combined additive and proportional errors. The inter-individual variability (IIV) model was described as a lognormal distribution.Pre-specified covariates were rationalized by their physiological relevance, and their effects on PK parameters were evaluated. BSA or body weight (BW), sex, age, and renal/hepatic function markers, including creatinine clearance (CrCL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, albumin, and total plasma protein, were tested for their effects on FCN-159 clearance. Cancer type was tested in relation to absorption parameters. BSA or BW, sex, age, red blood cell count, albumin, and total plasma protein were tested for their effects on volume of distribution.C; peripheral compartment, VP) using power functions. BW was normalized to 60 kg, and BSA to the median BSA value of 1.66 m2.C, or VP); θ either used empirical values  or were fitted for the adult data. The best allometric model was selected based on OFV, the plausibility of parameter estimates, and the precision of parameter estimates.To extrapolate the adult model for pediatric study simulation, allometric scaling based on body size was implemented. BW and BSA were tested as default covariates based on apparent clearance type  and volume of distributions . A non-diagonal variance-covariance matrix for IIVs was also evaluated. Forest plots were constructed by varying values for one covariate at a time to determine the effects of individual covariates on PK parameters and FCN-159 exposure. For forest plots, categorical covariates were assessed at each level, while continuous covariates were assessed at their reference values  and the corresponding 5th, 25th, 75th, and 95th percentiles.BSA as the allometric term was included in the model by default without statistical test as mentioned above. All other candidate covariates were screened by a univariate stepwise approach. In the first step, covariates that were potential predictors in the model were identified one by one. The covariate with the most significant reduction in the OFV along with chi-square test The stability of the model was verified by condition number . The model was considered stable with an eigenvalue of less than 1000. The robustness of the model and parameter estimates were assessed by the bootstrap method. Among 1000 bootstrap runs, a success rate greater than 80% was regarded as acceptable. Parameter estimates close to the median of bootstrap estimates suggests stable parameter estimates. The model’s predictability was verified by visual predictive check (VPC) . In the 2. The stochastic simulation included random resampling from IIVs and residual error distributions. IIVs and residual error distributions were assumed to be the same between adults and pediatric patients. The simulation was also assumed similar bioavailability between adults and children. The reference exposure range for adult patients was also simulated by random resampling from the actual subjects in the FCN-159-001 and FCN-159-002 studies.The developed adult population PK model was adapted using allometric scaling to inform pediatric dose selection in children aged 2–17 years. Simulations were performed to identify pediatric doses that would result in exposure comparable to the observed adult exposure at selected therapeutic dose of 8 mg. Virtual pediatric patients were simulated using a uniform BSA distribution between 0.55 and 2.15 mn = 500 in each band) and adult subjects (n = 500) were then simulated based on fixed integer dose levels . PK exposure metrics of steady-state areas under the curves  and maximum concentrations  were computed from the simulated PK profiles. The PK exposures  in children within each band were compared with the simulated adult reference exposures  using box plots. The BSA boundaries were further adjusted manually, and the final dosing bands were fine-tuned iteratively to achieve optimal matching to the adult exposure by visual inspection.Due to the constraints from the strength of the tablets , doses can only be administrated as an integer . Therefore, dosing bands were explored with the aim of balancing between 1) exposure variability within each band, and 2) minimizing the deviation from the theoretical optimal dose . Each dosing band had defined lower and upper boundaries for BSA, and all patients falling within that BSA range would get the same fixed dose. As expected, within each dosing band, the patients who are at the BSA boundary will have the largest deviation from their actual optimal doses. Therefore, a series of initial dosing bands are set as 0.5–1.5 mg, 1.5–2.5 mg, 2.5–3.5 mg and so on and the corresponding BSA boundaries were calculated accordingly using population mean exposure in adults and fixed effect’s PK parameters. Exposure for pediatric subjects  and −0.0134 (RSE: 54%), respectively. Therefore, those two covariates on clearance were removed from the final model. The removal of these two covariates resulted in slight increases of IIV in CL/F (from 17.2% to 19%) and OFV (from 2995.8 to 3014.724). The final covariate model included the effects of BSA on CL/F, VC/F, Q/F and VP/F; of total protein on VC/F; of sex on VP/F; and cancer type on F1. The final PK parameter and covariate relationships are presented in Besides BSA, other statistically significant covariates included in the model were: age, albumin on CL/F, total protein on V2 (corresponding to 5th and 95th percentiles of the population) would be expected to have 19% higher and 20% lower AUC0-24, ss and 31% higher and 29% lower Cmax, ss, respectively, relative to a typical subject with a BSA of 1.66 m2. The impacts of total protein and sex on AUC0-24, ss were minimal. However, a higher total protein would lead to a higher Cmax. For instance, a patient with total plasma protein of 76.89 g/L would be expected to have 30% higher Cmax, ss relative to a patient with total plasma protein of 68.95 g/L. Male patients would have approximately 3% higher Cmax, ss relative to female counterparts. The effects of each covariate on FCN-159 exposures  are illustrated in forest plots  are presented in vs. population predicted values and times from the first dose were randomly distributed around the line of identity and showed no tendency supporting the appropriateness of the structural and residual error models  were evaluated. Delayed absorption significantly improved the model’s fit. Compared with simple first-order absorption, none of the models with higher complexity provided significant improvement in terms of capturing the absorption profiles. Therefore, first-order absorption with first-order delay was chosen to describe the absorption phase of the FCN-159’s PK. Although the FCN-159-001 (melanoma patients) and FCN-159-002 (NF1 patients) studies used the same formulation, and higher exposure (both Cmax and AUC) was observed in the NF1 population. As Cmax is mainly driven by the volume of distribution whereas AUC is mainly impacted by clearance; when both parameters are impacted toward the same direction, a rational explanation is that bioavailability differed between two studies. Therefore, cancer type as a covariate was added to the bioavailability term in the model. The results indicated NF1 patients had roughly 30% higher bioavailability compared with melanoma patients, but the underlining mechanism remains unclear. One possibility for the lower absorption in melanoma patients may be the compromised gastrointestinal (GI) tract functions which is widely recognized for metastatic melanoma  class IV compound . The Tmelanoma . In contmelanoma  may be c2) = 0.1173 x BW (kg)0.6466. In this equation, BSA is only a function of body weight. Using this equation, exponent values of 0.75 and 1 on weight would translate to exponent values of 1.16 and 1.54 on BSA. The estimated exponents from the modeling dataset were 1.11 and 3.84 for apparent clearance and volume, respectively. The large difference between the estimated and empirically exponents for volume may be due to the limited BSA ranges included the modeling dataset. The current dataset contained BSA values ranging from 1.33 to 2.16 m2. The target population for the prediction had an estimated BSA range from 0.55 to 2.125 m2, which is much wider than the current modeling dataset. Nevertheless, a larger exponent, if overestimated, would result in an underestimation of volume of distribution prediction in young pediatric patients. As Cmax is affected by the volume of distribution and is mainly related to safety, a potential overestimation of Cmax would give a relatively conservative starting dose selection. In contrast, the estimated exponent for clearance is more consistent with the empirical value. Therefore, AUC prediction is likely to be more accurate than Cmax. When selecting the exposure metrics for matching, AUC was also chosen as the primary metrics because of the mode of the action of the drug. For FCN-159 to be effective, it requires constant inhibition of the target. The treatment for NF1 could last for years. For targeted antitumor therapy with chronic dosing, AUC is deemed more relevant than Cmax from efficacy perspective. For safety, the main adverse events (AEs) observed for FCN-159 including folliculitis and paronychia  and unlikely to be clinically meaningful. So, no dose adjustment based on total protein is deemed necessary.FCN-159 metabolism is mainly mediated by three CYP enzymes, including CYP3A4, CYP2C19 and CYP2C8. The expression and activities of these three CYP enzymes are expected to approach adult levels by 2 years of age. Thus, the organ maturation function was not included in the pediatric model . In addiSimulations were performed in the pediatric population with the goal of obtaining FCN-159 plasma exposure similar to that observed at the RP2D in NF1 adults . For dosOverall, the analyses described herein represent a comprehensive effort to use modeling and simulation approaches to extrapolate PK data of a MEK1/2 inhibitor from adults to pediatric patients for pediatric dose selection. FCN-159’s adult PK was described by a 2-compartment model with linear first-order elimination and first-order delayed absorption. Aside from the body size effect (BSA) on apparent clearance and volume and the patient’s effect on bioavailability, other covariates of interest, including sex, age, plasma protein and renal/hepatic function markers did not have clinically meaningful effects on FCN-159’s PK. Simulations from the final model were used to inform dose selection for pediatric dose development. BSA-based dosing bands were recommended, which is predicted to result in exposure that is comparable to the adult exposure at the clinically recommended dose.


Mollaret meningitis is a recurrent aseptic meningitis mostly caused by herpes simplex virus type 2. Other causes of the disease rarely exist, and its pathology is not well understood. Herein, we present a 57-year-old man who had been admitted to our hospital eight times with recurrent aseptic meningitis. Although the deoxyribonucleic acid (DNA) of varicella-zoster virus (VZV) was not detected in the cerebrospinal fluid (CSF), his genetic analysis, measurement of anti-VZV immunoglobulin-G (IgG) in the CSF, the VZV IgG index, IgG in the serum, and interleukin-1 beta in the CSF revealed that the Mollaret meningitis had been caused by the VZV. This case demonstrates that Mollaret meningitis can be caused by the VZV when specific factors are associated with decreased immune response. This case is valuable in elucidating the pathophysiology of Mollaret meningitis. Mollaret meningitis is a recurrent aseptic meningitis (RAM), which was first reported by Mollaret in 1944 . MollareThe pathology of Mollaret meningitis is not well understood. However, in recent years, immune disorders have been reported to be associated with disease onset ,5. We re3 without malignant or Mollaret cells, 74 mg/dL protein, and 62 mg/dL glucose, and an oligoclonal band was seen. We started treatment with acyclovir, and the fever went down and the headache improved. In the follow-up CSF evaluation, the pleocytosis had improved.In November 2020, a 56-year-old man was admitted to our hospital with the eighth recurrence of aseptic meningitis. During this recurrence, neurological findings showed only meningeal signs. No obvious rash or genital herpes was observed. No markers of an inflammatory response, including serum amyloid A, were observed in the serum. The cerebrospinal fluid (CSF) contained 205 mononuclear cells/mm3 cell count at the extreme stage of recurrences, and improvements in CSF findings can be obtained along with improvements in clinical symptoms. Mollaret's cell was detected in the seventh recurrence  has been detected in the CSF to date. The patient had always visited the hospital with headache and fever for several days, and only signs of meningeal irritation were observed as physical findings. The CSF contained about 200 mononuclear cells/mme Figure . CranialDuring the seventh and eighth recurrences, anti-VZV immunoglobulin-G (IgG) (enzyme immunoassay) in the CSF increased by 0.7 and 0.9, respectively , while the VZV IgG index (CSF virus-specific IgG titer/serum titer) increased by 3.56 and 0.9, respectively   and GaldWe performed several additional analyses. Human immunodeficiency virus-1/-2 antigen and antibody screening tests were negative. The HLA haplotypes of the patient were HLA-B7 (07:02:01)/B52 (52:01:01) and HLA-DR15 (15:02:01)/DR1 (01:01:01). Correspondingly, the patient showed mildly decreased serum IgG (690 mg/dL). IL-1β was not detected in any CSF sample  inflammasome and the associated processing of proinflammatory cytokine IL-1 by activated caspase-1 in infected cells . FurtherInitially, the patient was also suspected of having Mollaret’s meningitis due to FMF. The E148Q mutation is believed to be a benign polymorphism and not a disease-causing mutation . Capron Although cases in which IL-1β was measured in the CSF of FMF patients have not been reported, IL-1β in CSF increased in patients with CNS neuropsychiatric systemic lupus erythematosus and infections of the CNS compared to healthy controls , and itsThe pathology of Mollaret meningitis, including causative microorganisms, has not been fully clarified. We consider that the dysfunction, probably due to genetic factors, of multiple immune pathways against infectious viruses affecting the CNS underlies the pathogenesis of the disease. We observe that VZV can be a pathogenic microorganism of Mollaret meningitis. We hope that the pathology of this disease will be elucidated and that appropriate treatment will be established.


A 23-gene classifier, RecurIndex® , has been developed based on the gene expression profiles of early-stage breast cancer patients of ethnic Han Chinese population in Taiwan. This study aimed to compare the performance of the Oncotype DX® RS with the RecurIndex® recurrence index (RI) for predicting relapse-free survival. Therefore, we calculated both the RI and RS for 110 early stage breast cancer patients, with the cut-off value for high-risk recurrence set at 26 and 29 for the RS and the RI, respectively. With relapse-free interval (RFI) as the primary endpoint, the concordance between RS and RI was 78.2% . For a median follow-up interval of 27 months, there was a statistically significant difference in RFI between the high- and low-risk groups defined by the RI (p = 0.04) but not between risk groups defined by the RS (p = 0.66). In conclusion, whereas there was high concordance between the RecurIndex® RI and the Oncotype DX RS, the current data showed that the RI had a better discrimination for recurrence risk than the RS. Subsequent studies with larger sample sizes will be needed to confirm the superiority of the RI over the RS in the Asian population. The applicability of the Oncotype DX Among noutcomes .®, EndoPredict® , and MammaPrint® , can help guide treatment decisions. However, these commercially available assays have been developed based on the genomics of Western populations [Genomic tests for breast cancer prognosis, such as Oncotype DXulations . The inculations . For exaulations . In contulations . Hence, ulations ,11.®, was initially developed using the microarray platform technology [® [® in the Taiwanese population and compared the prognostic performance of the test with the 23-gene classifier using the same patients’ data obtained from the RT-PCR platform. The 23-gene classifier, RecurIndexchnology ,11,12 toology [® . However® test results from three medical centers in Taiwan, were recruited to participate in this study and had their formalin-fixed, paraffin-embedded (FFPE) tumor tissue sections tested with the 23-gene classifier. A total of 110 patients with the following inclusion criteria were included in this study  FemThe development of the 23-gene classifier has been described previously ,11,12. T® RS, we defined the risk groups as in the TAILORx trial, with a recurrence score (RS) < 25 indicating non-high-risk, and scores ≥ 25 indicating high-risk. For the risk determination based on data from the RT-PCR platform, the cut-off value for the 23-gene classifier was set to 29, using the receiver operating characteristic (ROC) analysis to determine the best combination of sensitivity and specificity. Patients with the 23-gene classifier recurrence index (RI) ≥ 29 were defined as having high-risk of recurrence, and those with 23-gene classifier RI < 29 were defined as low risk. For the Oncotype DX® RS for predicting the actual status of recurrence in each patient within five years after surgery. In addition, a Kaplan–Meier plot was used to observe the survival time, with the log-rank test used to compare the relapse-free interval (RFI) between the high- and low-risk groups as determined by the two assays. Furthermore, we calculated Cohen’s kappa coefficient to evaluate the agreement between the Oncotype DX® RS and the 23-gene classifier RI.The clinical performance of the two assays was evaluated by calculating the negative predictive value (NPV), positive predictive value (PPV), sensitivity, and specificity of the 23-gene classifier and the Oncotype DXp = 0.046). The distribution of tumor grade was nearly statistically significantly between the low- and high-risk groups of patients (p = 0.068).®. Regarding the tumor grade, there were significantly more grade III in the high-risk group (25.00%) than in the low-risk group . In addition, among the only two patients that had recurrence, one was in the high-risk whereas one was in the low-risk group.In ® RS for early breast cancer patients. Only one (66.67%) of the two recurrence cases were classified into the high-risk group (with a PPV of 5.6%), whereas one recurrence case was classified into the low-risk group (with an NPV of 98.9%).® RS and the 23-gene classifier RI. Among the 110 patients, 24 (22%) and 86 (78%) were categorized by the 23-gene classifier RI as high- and low-risk, respectively, whereas 16 (15%) and 94 (85%) were classified by the Oncotype DX® RS as high- and non-high-risk, respectively. For a total of 88 patients (80%), the two assays agreed: 9 (8.2%) into the high-risk and 79 (72%) into the low/non-high-risk groups as determined by both the Oncotype DX® RS and the 23-gene classifier RI. For concordance between the 23-gene classifier RI and the Oncotype DX® RS, the Cohen’s kappa coefficient was 0.297, indicating a low concordance between the two assays. As shown in ® RS .p = 0.04). The 7-year RFI for the low-risk group was 100%, whereas that for the high-risk group was 61% (interquartile range 27–100%). In comparison, for the Oncotype DX® RS, the 7-year RFI for the low-risk group was 98% (93–100%) and that for the high-risk group was 75% (43–100%) (p = 0.66). In ® assay has shown that patients with breast cancer of the luminal subtype  and with node-negative status can be spared from adjuvant chemotherapy [It has been shown that most of the early stage breast cancer patients of the luminal subtype remained non-recurrent even when the patients were not treated with adjuvant chemotherapy . It woulotherapy . ® assay and other breast cancer prognostic assays on the market were developed based on the Western populations and may not be suitable for the Asian population [® test may overestimate recurrence risk of Asian populations. In addition, our preliminary analysis in 44 Belgium patients of GSE45255 [® and the genetic model of RecurIndex®. There were 35 high-risk subjects determined by Oncotype DX®, whereas 11 high-risk subjects were determined by RecurIndex®, which shows that the prognostic capability depends on the target population of the prognostic tool. The details of the results are shown in the ®, a 23-gene classifier, was thus developed based on the ethnic Han Chinese breast cancer population in Taiwan to meet unmet clinical needs [However, the Oncotype DXpulation . In factpulation . More sppulation , suggestGSE45255  showed dal needs .® and the Oncotype DX® assays and compared them against each other for correlation and concordance. The 23-gene classifier for recurrence prognosis of breast cancer showed good performance in predicting the outcomes of any recurrence within 5 years after surgery since all of the 86 patients classified as low-risk by the assay were free of relapse during the 5-year follow-up period, with the NPV determined to be 100%  and concordance (Cohen’s kappa = 0.297) between the two assays (® assay may have potential in providing prognostic information on breast cancer for patients other than ethnic Han Chinese. Further studies will be needed to reveal such potentials.In contrast, the Oncotype DXo assays , suggestIt should be noted that there are some limitations in this study. Firstly, most of the participants in this study had the luminal subtype of breast cancer. This subtype has a relatively good prognosis when compared to other subtypes, but it is also associated with late recurrence, i.e., recurrence in more than five years after receiving the primary surgery treatment . Since t® assay, which was developed based on the Asian population, may be used to identify low-risk breast cancer patients of Asian descendant. To further validate the performance of the 23-gene classifier, a larger cohort and a longer follow-up will be needed.In summary, the current study showed that the 23-gene classifier, RecurIndex


Plasmodium knowlesi (Pk-ClpP) and the inhibition properties of a δ-lactone hyptolide against this protein. Accordingly, a codon-optimized synthetic gene encoding Pk-ClpP was expressed in Escherichia coli BL21(DE3) and purified under a single step of Ni2+-affinity chromatography, yielding a 2.20 mg from 1 L culture. Meanwhile, size-exclusion chromatography indicated that Pk-ClpP migrated primarily as homoheptameric with a size of 205 kDa. The specific activity of pure Pk-ClpP was 0.73 U µg−1, with a catalytic efficiency kcat/KM of 0.05 µM−1 s−1, with optimum temperature and pH of 50 °C and 7.0–7.5, respectively. Interestingly, hyptolide, a member of δ-lactone, was shown to inhibit Pk-ClpP with an IC50 value of 17.36 ± 1.44 nM. Structural homology modelling, secondary structure prediction, and far-UV CD spectra revealed that helical structures dominate this protein. In addition, the structural homology modeling showed that this protein forms a barrel-shaped homoheptamer. Docking simulation revealed that the inhibition was found to be a competitive inhibition in which hyptolide was able to dock into the catalytic site and block the substrate. The competitiveness of hyptolide is due to the higher binding affinity of this molecule than the substrate.The caseinolytic protease (Clp) system plays an essential role in the protein homeostasis of the malaria parasite, particularly at the stage of apicoplast development. The inhibition of this protein is known to have a lethal effect on the parasite and is therefore considered an interesting avenue for antimalaria drugs discovery. The catalytic activity of the Clp system is modulated by its proteolytic subunit (ClpP), which belongs to the serine protease family member and is therefore extensively studied for further inhibitors development. Among many inhibitors, the group of β-lactone is known to be a specific inhibitor for ClpP. Nevertheless, other groups of lactones have never been studied. This study aims to characterize the catalytic properties of ClpP of  Plasmodium knowlesi is being increasingly recognized as an important contributor to malaria infections in Southeast Asia, including Malaysia, Myanmar, and Indonesia  was then converted into a fraction unfolded (%) and fitted to get the Tm value according to Tripathi  value at 222 nm were monitored upon the gradual increment  was prepared in ethanol. For specific activity measurement, the reaction cocktail included 10 µM of the substrate in 0.1 mM Tris-HCl buffer, pH 8.0. Various concentrations of Pk-ClpP were then added to the cocktail and incubated for 10 min at 25 °C. The amount of p-nitrophenol released was calculated based on an extinction coefficient for p-nitrophenol of 18,000 M−1 cm−1 at 340 nm using Lambda 25 UV-Vis Spectrophotometer . One unit activity is defined as the amount of protein needed to produce 1 µmol of p-nitrophenol every minute. The kinetic parameters of the Pk-ClpP were characterized in terms of Michaelis–Menten kinetic parameters  using non-linear regression with GraphPad Prism V.8 [The catalytic activity of Pk-ClpP was determined based on the method in El-Bakkouri et al.  with sevrism V.8 . For thip-nitrophenyl as a substrate by following the condition as described above. To determine the pH profile of the enzyme, the activity was performed in a pH range of 3 to 11 using 50 mM acetate (pH 3.6–5.6), 50 mM phosphate (pH 5.8–8.0), and 50 mM glycine (pH 8.6–10.6) buffers. All assays were performed in triplicate.The experiment was performed based on Razali et al. . The effp-nitrophenyl in the presence of various concentrations of hyptolide. The hyptolide was prepared from Hyptis pectinata (L.) as previously described [50 value, which refers to the concentration of sample required to reduce 50% of the catalytic activity, was calculated using a four-parameter logistic curve under the SigmaPlot 14.0 statistical software . The assay was performed in triplicate with a serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) as a control.The effect of hyptolide on the catalytic activity of Pk-ClpP was determined by measuring the catalytic activity against N-CBZ-Glycine escribed  and dissLigand structures (hyptolide and substrate) were built using Discovery Studio by inserting the SMILE format and generating a 3D structure with the chemical builder. The 3D structure of Pk-ClpP was built based on the homology modeling technique. The main step is searching for the best template, alignment, and structure building. We employed the SWISS-MODEL server as standard protein modeling. The Pk-ClpP model was validated based on the global model quality estimation score (GMQE). Both ligands and protein were prepared in the MOE software. Preparation was conducted for 3D protonation to add missing hydrogen atoms to the protein and ligand structure. Three-dimensional structures were corrected for the unfavorable steric clash. Finally, prepared Pk-ClpP, hyptolide, and substrate were ready for the docking process.PyRx molecular docking software was used to predict the competitive binding of inhibitors on the target site. The binding site of Pk-ClpP was determined manually by selecting triad catalytic site . Docking protocols used the Autodock Vina scoring function and Lamarckian genetic algorithm. Nine docking results of hyptolide were evaluated, and the best docking score was compared to the native ligand (substrate). All results were analyzed using the MOE software to determine the molecular interaction.This study is considered an initial insight into the functional properties of Pk-ClpP, about which the following conclusions may be drawn. Firstly, Pk-ClpP was found to be catalytically active, albeit with lower activity than Pf- and bacterial ClpPs. Secondly, the lower similarity of Pk-ClpP to human ClpP suggests that targeting Pk-ClpP should have a low risk of mistargeting host ClpP. Thirdly, Pk-ClpP and Pf-ClpP share similar oligomeric structures in solution (homoheptameric). Lastly, the inhibition activity of the δ-lactone-based molecule demonstrated in this study provides an interesting avenue to further explore this compound for novel antimalarial drugs.


Mechanistic studies revealed that the clearance of senescent cells was mediated by the activation of the Fas signaling pathway. The in vivo injection of chiral NPs successfully confirmed that the elimination of senescent microglia cells in the brain could further alleviate the symptoms of PD mice in which the alpha-synuclein (α-syn) in cerebrospinal fluid (CFS) decreased from 83.83 ± 4.76 ng mL−1 to 8.66 ± 1.79 ng mL−1 after two months of treatment. Our findings suggest a potential strategy to selectively eliminate senescent cells using chiral nanomaterials and offer a promising strategy for alleviating PD.Parkinson's disease (PD) is an age-related neurodegenerative disease, and the removal of senescent cells has been proved to be beneficial for improving age-associated pathologies in neurodegeneration disease. In this study, chiral gold nanoparticles (NPs) with different helical directions were synthesized to selectively induce the apoptosis of senescent cells under light illumination. By modifying anti-B2MG and anti-DCR2 antibodies, senescent microglia cells could be cleared by chiral NPs without damaging the activities of normal cells under illumination. Notably,  The apoptosis pathways of senescent microglia cells induced by chiral NPs under the irradiation of 808 nm laser in the brain of PD mice. It had been reported that senescent cells could give rise to local and systemic inflammation and contribute to neurodegeneration in neurodegeneration diseases like PD.20–22 In addition, direct exposure to Aβ was shown to cause senescence in oligodendrocyte precursor cells (OPCs), and the clearance of OPCs by senolytic therapy alleviated Aβ-associated inflammation and restored cognitive deficits in AD mice, thus illustrating the potential for senescence clearance to be used in clinical practice.23 Moreover, senescent cells play a role in the initiation and progression of tau-mediated disease, and targeting of senescent cells may provide a therapeutic avenue for the treatment of such pathologies.24 Therefore, eliminating senescent cells may hold therapeutic promise for alleviating the symptoms of PD.25Parkinson's disease (PD) is an age-related brain disease that is associated with motivation and cognitive disorders and the assembly of alpha-synuclein (α-syn); there is no effective therapeutic treatments for this condition.26–28 The different interactions of chiral nanoparticles (NPs) with bio-interfaces can induce a range of biological reactions.27,29,30 Our group has made several key breakthroughs in this field. For example, we found that the maturation of immune cells can be regulated by nanoscale chirality via differential affinities for biological receptors.31 Moreover, due to their biocompatibility and easily controllable optical activity, chiral NPs have already been applied for the elimination of senescent cells and the restoration of homeostasis.13,32 Light provides a non-invasive method for controlling these light-responsive nanomaterials due to its precise spatial resolution, appropriate controllability and the lack of negative effects in these light-responded biomimetic systems consisting of nanomaterials.26,33–35 Alternatively, a recent study has demonstrated that one biomimetic NP could effectively target and modulate microglia cells and thus be expected to be a potential medicine against PD.36 Collectively, the evidence provides the possibility of alleviating neurodegenerative disease by the application of chiral NPs to clear senescent microglia cells.37–39Inorganic nanomaterials that exhibit chirality exist commonly in nature and have attracted significant attention due to their promising properties and potential for multi-biological applications related to the adjustment of life progression.l-P+ NPs exhibited the maximum clearance efficiency of senescent cells without damaging normal cells. Senescent microglia cells were eliminated by the activation of an apoptotic pathway by mechanical force under light; this was demonstrated by the increased expression of apoptotic genes in the treatment groups. We also used a mouse model of PD to evaluate this potential therapeutic strategy in vivo. Following two months of treatment, the pathological symptoms of the PD mice were greatly improved with the decreased concentration of α-syn and the repair in motor ability.In the present study, we prepared chiral NPs with high anisotropy factors to eliminate senescent microglia cells from the brain under NIR irradiation at 808 nm. We found that l-P+ NP, d-P− NP, and dl-NP) of different handedness were synthesized in accordance with a previous study using l-, d-, and dl-Cys-Phe (CF) as the chiral ligands.31 As shown in Fig. S1a and b,dl-NPs with no chiral morphology are shown in Fig. S1c.g-factor) of l-P+ NPs reached 0.44 at 711 nm, while that of d-P− NP reached 0.42 at 722 nm, and that of dl-NP was 0  for 7 days to induce senescent models (DOX-Sen) to monitor the pathological symptoms in the brains affected by PD.33 After incubation with DOX, the increased expression of β-galactosidase  and up-regulated expression of p16INK4a (a known marker of senescence)13 confirmed the successful construction of the senescence models in vitro (-1β and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA) to indicate the state of inflammation in the senescent cells. When incubated with DOX, the BV-2 cells showed signs of senescence with increased expression levels of inflammation markers.A previous study had illustrated that microglia cells are associated with the loss of dopamine neurons, which was considered as one of the main reasons for PD.in vitro . In addil-P+ NPs. Therefore, we used 0.8 nM of anti-DCR2 and 1 nM of anti-B2MG for the subsequent experiments as we had optimized in experimental cells. In addition, to evaluate the difference of nanomaterials with different chiral morphologies in binding antibodies, the CD spectrum in 200–280 nm was collected. As shown in Fig. S6,l-P+ NP, d-P− NP, and dl-NP after removing the dissociative antibodies.Then, the appropriate concentrations of anti-DCR2 and anti-B2MG binding to the NPs were optimized under two-photon luminescence (TPL) after incubating the chiral NPs with senescent BV-2 cells. As shown in Fig. S4 and S5,−1 exerted no obvious cytotoxic effects on the three types of senescent cells. Furthermore, to illustrate the chiral effect of NPs on senescent cells, we investigated the uptake ability of senescent BV-2 cells for chiral NPs. The relationship between the incubation time and the amount of intracellular chiral NPs was determined from the TPL intensity  to clarify it  experiments to monitor the cytotoxicity of chiral NPs in senescent cells Fig. S7. We founntensity  and S8†.nt cells  and S9†.l-P+ NPs with or without antibodies coated were taken in different cells, normal and senescent BV-2 cells were incubated with different inhibitors of endocytosis pathways.31 As shown in Fig. S15 and S16,l-P+ NP@anti-DCR2@anti-B2MG in both normal and senescent BV-2 cells was inhibited when the pathways of clathrin and dynamin were blocked respectively. Simultaneously, the uptake of l-P+ NP in normal and senescent cells was studied  and control groups (senescent BV-2 cells without any treatment), as summarized in Plaur, Mmp12, Il1b, Ccl5)7,15,16 was significantly reduced in senescent BV-2 cells after treatment with l-P+ NP under light. Moreover, as compared with the control group, the expression levels of anti-apoptotic genes 13 were decreased while the levels of proapoptotic genes  were increased in senescent BV-2 cells by l-P+ NPs under light.41 Importantly, we found that in the senescent microglia cells treated with l-P+ NPs under light, the expression levels of Fas signaling pathway-associated genes ,42,43 an outcome mediated by the activation of the apoptosis pathway, were higher than in any of the other groups. These data indicated that the apoptosis of senescent cells induced by l-P+ NPs was through the Fas signaling pathway.Next, we investigated the global expression profiles of microglia cells to determine the mechanisms responsible for the induction of apoptosis in senescent cells by chiral NPs under light . There w44–46 the expression of cleaved caspase-8, the key protein in this pathway, was measured. As shown in d-/l-NP and light treated cells was higher than that in the control, of which the expression in l-P+ NPs was 22.09-fold higher than that in the control and that of d-P− NPs was 12.21-fold higher. This result clearly confirmed that apoptosis in chiral material treated groups was mediated by the Fas signaling pathway. Notably, the expression of caspase-8 in the group treated with l-P+ NPs and light was 1.81-fold higher than that with d-P− NPs and light , were injected into the striatum of each mouse 4  were used for the .88 mm)4 .INK4a+ and MAP2+; senescent astrocytes: p16INK4a+ and GFAP+; senescent microglia p16INK4a+ and IBA-1+), l-P+ NPs labeled with Cy3 in different brain cells were measured by flow cytometry. As shown in Fig. S34,l-P+ NPs were in senescent cells. In addition, 78.2% of the Cy3 fluorescence was detected in senescent microglia cells, which indicated that the l-P+ NPs were almost taken up by senescent microglia cells  in the brain of PD mice. We found that the expression of microglia and p16INK4a overlapped obviously while the expression of markers for neurons and astrocytes did not overlap with senescence markers , a microglia marker (IBA-1), an astrocyte markers (GFAP), and a senescence marker . The reduced amount of α-syn in the treated group was indicative of potential remission of PD after treatment with chiral NPs under irradiation for 60 days. l-P+ NPs and light while that of α-syn decreased. Corresponding β-Gal staining images of the nigra also indicated the clearance of senescence in PD mice treated with l-P+ NPs under light  to detect TH and α-syn  and S50†In vivo mice experiments demonstrated that l-P+ NPs accumulated in senescent microglia cells in the brain of PD mice and effectively cleared senescent microglia cells under light, which decreased the amount of α-syn in CSF and recovered the dyskinesia of PD mice. This discovery paved the way for the use of chiral nanomaterials for fundamental research in clinical neurodegeneration disease therapy.In conclusion, we successfully fabricated light-responsive chiral gold NPs for the clearance of senescent microglia cells selectively. All data supporting the findings of this study are available within the article and in the ESI.†C. X., H. K., and M. S. conceived the concept and directed the project. Z. X. and A. Q. carried out the experiments and analysed the data. W. W., M. L. and B. S. collected the data. C. H., H. Z. and L. X. discussed the results. Z. X. prepared the first draft of this manuscript, and M. S. and C. X. modified the manuscript. All authors commented on the manuscript.The authors declare no competing financial interest.SC-013-D2SC01662A-s001